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Characterization of ET1 ep-/- mouse line

Characterization of ET1 ep-/- mouse line. By Steven Ma Mentor: Dr. Arup Indra Pharmaceutical Sciences Sep 25, 2009. Why ET1 (endothelin-1)?. Known growth factor – hyperproliferative pathologies: psoriasis and cancer?.

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Characterization of ET1 ep-/- mouse line

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  1. Characterization of ET1ep-/-mouse line By Steven Ma Mentor: Dr. Arup Indra Pharmaceutical Sciences Sep 25, 2009

  2. Why ET1 (endothelin-1)? • Known growth factor – hyperproliferative pathologies: psoriasis and cancer? • Endothelin “axis” has been shown to promote cancer progression (Bagnato et al. 2008) • Melanoma • Breast, prostate, brain, lung, bladder etc. • Malignant melanoma is the leading cause of death (75%) in skin cancers - no curative measures yet (Jerant et al. 2000)

  3. Endothelins What we know: • 21 amino acid peptides • Three isoforms, ET-1, ET-2, ET-3 • Produced by keratinocytes at a basal level • What we don’t know: • Autocrine functions in keratinocytes http://upload.wikimedia.org/wikipedia/commons/a/ac/1EDP_human_endothelin1_03.png

  4. Overview of Skin • Many Layers • 95% are keratinocytes • Melanocytes • Secrete melanin – responsible for pigmentation (and tanning) • Derived from neural crest cells (same as neurons) Diferentiation Proliferation

  5. Hypothesis ET1 has an autocrine effect on epidermal keratinocyte homeostasis, altering cell proliferation and differentiation. ET1 has a paracrineeffect on melanocytes.

  6. Getting ET-1 out of epidermis • Bacteriophage P1 site-specific Cre-lox recombination • Flank target genes with loxP sites • Cre – Cyclic Recombinase • Human – mice homology x http://upload.wikimedia.org/wikipedia/commons/5/58/CreLoxP_experiment.png

  7. Techniques • PCR: to confirm ET-1 ablation in epidermis • Paraffin sections of skin treated with 4% PFA (paraformaldehyde) used for: • Hematoxylin and eosin staining • Immunohistochemistry • Fontana Masson staining

  8. Hematoxylin & eosin staining (HE) • Stains nucleus blue and cytoplasm pink • Popular method in histology, widely used in medical diagnosis, e.g. tumor biopsies • Used to measure epidermal thickness

  9. Data: epidermal thickness Epidermal thickness (µm) No significance E{ E{ ET1L2/L2 (wildtype) D D HF HF HF HF ET1L2/L2 (wildtype) ET1ep-/- ET1ep-/- (knockout)

  10. Immunohistochemistry (IHC - with paraffin sections) • Use antibodies to detect specific proteins in skin sections • Purpose: analyze cell differentiation and proliferation • Used Cy3 2ndary antibody

  11. Epidermal Proteins Loricrin Late differentiating cells Keratin 10 (K10) Late differentiating cells Keratin 14 (K14) Early proliferating cells Ki 67 Proliferation marker; present in all active cell phases (but not G0)

  12. Immunohistochemistry – differentiation Red – Cy3 Blue – Dapi (all cells) ET1L2/L2 (Wildtype) ET1ep-/- K14 E D K10 E D Loricrin E D

  13. Immunohistochemistry – proliferation Red – Cy3 Blue – Dapi (all cells) ET1L2/L2 (Wildtype) ET1ep-/- E D Ki 67 Counts (per 100 cells):

  14. Fontana-Masson Staining (FM) • Stains all nucleired, but melanocytesblack • “Melanin stain” • Used to analyze paracrine effect of • keratinocyte ET-1 on neighboring melanocytes

  15. Fontana-Masson Stain ET1L2/L2 (Wildtype) ET1ep-/-

  16. Fontana-Masson Stain:melanocyte count compared

  17. Results thus far • No significant autocrine effect on keratinocytes: • Proliferation • differentiation • Significant effect on melanocyte proliferation • Adult mice

  18. Ongoing experiments • UV treatment to new born mice • Skin samples at 0h, 24h, 48h time points • Fontana-Masson staining analysis • qPCR for melanogensis factors in UV treated mice

  19. Fontana-Masson Stain: UV treatment – 0 hour time point

  20. Special thanks to: Dr. Kevin Ahern Dr. Arup Indra Steven Hyter Dr. GitaliIndra Xiaobo Liang Gaurav Bajaj DanColemanZhixing Wang

  21. References • Jerant, A; Johnson, J; Sheridan, C; Cafferey, T. “Early Detection and Treatment of Skin Cancer.” American Family Physician. July 15, 2000 • Bagnato, A; Spinella, F; Rosano, L. “The endothelin axis in cancer: the promise and the challenges of molecularly targeted therapy. Can J PhysiolPharmacol. August, 2008. p473-84

  22. Questions?

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